Cholesteatomas are epithelial tumor-like growths that arise from the tympanic membrane, typically as a sequela of chronic otitis media. The progressive nature of this lesion commonly results in the destruction of middle and inner ear structures via osteoclastic bone resorption. This destruction leads to hearing loss, vestibular dysfunction and intracranial complications. Current management of this disease is limited to surgical eradication, which must often be repeated. Chronic infection is another hallmark of the cholesteatoma and we have therefore been interested in the inflammatory signaling pathways that lead to osteoclast activation. We recently established a role for nitric oxide synthase 1 (NOS1 or neuronal NOS) in osteoclast activation. We now wish to explore this role further by identifying the mechanisms by which NOS1 is activated and the endogenous modulators that keep nitric oxide production in check. To this end, we have outlined the following specific aims. Specific Aim 1: To localize the cellular source(s) of NOSl that mediate osteoclast activity and to also localize potential endogenous modulators of NOSl. Specific Aim 2: To identify the mechanism of activation for NOSl and assess the efficiency by which various modulators can control nitric oxide production and ultimately the bone resorbing activity of osteoclasts in models of cholesteatoma. Neurons control NOSl activation by functionally linking it to the NMDA type of glutamate receptor. Interestingly, osteoclasts also have functional glutamate receptors, including the NMDA subtype, but their function is not well understood. Further, glutamate receptor antagonists inhibit the ability of the osteoclast to seal to the bone, a prerequisite to resorption activity. Osteoclasts from mice with targeted deletions of the NOS1 gene exhibit a similar phenotype. This coincidence offers intriguing evidence that these two observations are related and that osteoclasts may use the same mechanisms that neurons do to modulate NOS1 activation. These studies offer important insight into the control of osteoclast activity in cholesteatoma and may lead to the development of pharmacologic interventions that would replace the need for surgery in the treatment of this debilitating disease.